This proposal is to perform preliminary studies that will ultimately result in antibiotics with synergistic activity against the HTV opportunistic pathogen, Mycobacterium tuberculosis (TB). TB is the leading cause of death among HTV-infected patients in the developing world. This bacterium predictably becomes resistant to each drug soon after it is introduced. A new approach is required that specifically prevents the emergence of drug resistance and its transmission between patients. We have discovered that mutations in codon 306 of the TB embB gene predisposes strains to acquire progressively more drug resistance and increases their potential for transmission, although the mutation does not cause resistance to any given drug on its own. Here, we propose to determine the mechanisms underlying these observations, with the ultimate goal of designing drugs that inhibit this activity. We hypothesize that the drug ethambutol (EMB) normally acts synergistically with other antibiotics to prevent the emergence of drug resistance, and that this synergy is lost in embB306 mutants. This synergy appears to be effective below the minimal inhibitory concentration of EMB. To better understand this process, we will investigate whether EMB-associated inhibition of lipoarabinomannam (LAM) biosynthesis increases the permeability of the TB cell wall to antibiotics and results in synergy. We will also investigate whether increased intracellular antibiotics can delay the development of drug resistance. Finally we will study whether EMB-inhibition of LAM modulates the early interactions between TB and the immune system, potentially explaining the increased transmissibility seen in the embB306 mutants. Experiments with wild type and embB3Q6 mutant strains will be performed in parallel to determine if embB306 mutants are resistant to these novel effects of EMB. Our specific aims are to determine in vitro: 1. if EMB causes synergistic with other antibiotics by increasing antibiotic permeability, and if embB306 mutants are resistant to this synergy; 2. if embB306 mutants have altered phagocytosis, growth and cytokine induction in macrophages and dendritic cells with and without EMB treatment; 3. if embB306 mutants are more likely than wild type strains to become resistant to other drugs in vitro; 4. if embB306 mutations affect biosynthesis of LAM, capped LAM, arabinogalactan (AG) or other cell wall components in the presence and absence of EMB.